Meningococcal oligosaccharide linked polysaccharides and diptheria protein conjugate vaccine for all ages

ABSTRACT

Methods for producing quadrivalent meningococcal meningitis polysaccharide and conjugate vaccines for serotypes A, C, Y and W-135 disclosed.  Neisseria meningitidis  fastidious medium was designed to maximize the yield of capsular polysaccharides and generate minimal cellular biomass and endotoxin in a short duration of fermentation. The crude polysaccharides are isolated, purified, and mechanically depolymerized by sonication. These purified polysaccharides were found in human clinical trials to be safe and immunogenic against meningococcal disease caused by  N. meningitidis  A, C, Y and W-135 serogroups in sub-Saharan Africa. In the preferred embodiment, the polysaccharides are conjugated to carrier proteins of diphtheria or tetanus toxiod to an average molecular size of 5100 to 9900 Daltons and provide broad spectrum protection to humans of all ages. Accelerated polysaccharide production and the efficacy of the resulting vaccine are demonstrated.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. Ser. No.11/680,471, filed Feb. 28, 2007, now based on the U.S. Pat. No.7,491,517 which was filed as a provisional application U.S. Ser. No.60/831,682 filed on Jul. 19, 2006.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to the field of medicalmicrobiology and immunology. In particular, the invention pertains to animproved vaccine for immunization.

2. Description of the Related Art

Neisseria meningitidis (the meningococcus), identified in 1887 is one ofthe causative agents of meningococcal meningitis. Twelve subtypes orserogroups of N. meningitidis have been identified and four (N.meningitidis. A, B, C and W-135) are known to cause epidemics. The mostcommon symptoms are stiff neck, high fever, sensitivity to light,confusion, headaches, and vomiting. Even when the disease is diagnosedearly and adequate therapy instituted, 5% to 10% of patients die,typically within 24-48 hours of the onset of symptoms. The 10 to 20% ofthe survivors suffer brain damage, hearing loss, or learning disability.A less common but more severe (often fatal) form of meningococcaldisease is meningococcal septicaemia which is characterized by ahaemorrhagic rash and rapid circulatory collapse.

Meningococcal bacteria incorporate polysaccharides into their surfacestructure. Thus a large majority of bacteria are covered with of capsuleor glyco-calyx polysaccharide which induces an immunological response inhumans. The outer membrane of gram-negative Neisseria meningitidis (NM)bacterium consists, inter alia, of lipopolysaccharide (LPS). Suchpolysaccharides (PS) are formed on the basis of repeating units in whichthe constituents and the bonds are defined and which are eachcharacteristics of the NM serogroups defined. These repeating unitscontain the epitopes which are the antigenicity-determining structures.

The immunogenicity of the capsular polysaccharides to carrier proteincan be improved by coupling them to Lipo-oligosaccharides (LOS). Whencovalently linked to a carrier protein, the resulting PS component in aconjugate vaccine becomes a T cell-dependent (TD) antigen inducinglong-term immunity with immune memory even in infants and youngchildren. Further, additional linkage with oligosaccharide (OS) makes arobust conjugate vaccine since OS not only acts as additional immunogenbut also as a vaccine adjuvant.

EXISTING STATE OF THE ART

The existing state of art has many problems as discussed. Precipitatingpolysaccharides with phenolic extraction for removing components(impurities) like lipopolysaccharide endotoxin results in phenoliccontaminants interfering with the pure polysaccharide productionprocess. Similarly, precipitating with catalyon and depolymerisedchemical hydrolysis also have problems like depolymerization interferingwith purity in vaccine production.

Existing art for depolymerization of polysaccharide by chemical meansand conjugation of polysaccharides with carrier proteins activated bychemical means state that the chemical residues tend to induce adverseside effects during routine immunization or the average size of thepolysaccharides obtained may not provide efficient immune response inhumans. Similarly, existing art regarding the inclusion of adjuvant forenhancing immunogenicity against the different serotypes of N.meningitidis may have adverse side effects during routine immunization.

Large scale biomass production with reduced production of capsularpolysaccharides is found in the prior art. Existing art on animal freemeningococcal polysaccharide fermentation medium with soy peptone asnitrogen source requires pH adjustment during fermentation. Also thehigh glucose utilization in the medium results in excessive cellularbiomass.

The current vaccines against this pathogen (Neisseria meningitidis) havethe following disadvantage such as the serogroups A, C, Y, and W-135capsular plain polysaccharide and the capsular polysaccharide diphtheriaconjugate vaccine are not for all ages and less immunogenic in youngchildren, especially infants.

Therefore there is a need for an invention to eliminate theshort-comings in the prior art and to invent a method of producing ameningococcal meningitis vaccine without any chemical impurities orresidues (to eliminate the disadvantages regarding depolymerization andconjugation by chemical means and capsular polysaccharide size) and amedium for producing it, which ensures higher yield of polysaccharidesand lower yield of cellular biomass to facilitate the production andpurification processes of vaccine production. Also there is a need foran improved meningococcal vaccine to prevent meningitidis deaths intoddlers and be a vaccine for all ages. Therefore it is an object of thepresent invention to invent a method of producing meningococcalmeningitis vaccine comprising N. meningitidis serotypes A, C, Y andW-135 having long lasting effect and provide broad spectrum immunity tohumans of all age groups.

It is yet another object of the present invention to develop a methodwherein trace chemical impurities currently present in the availablemeningococcal meningitis vaccine are eliminated by a mechanical method,preferably sonication.

Another object of the present invention is to invent a composition of amedium that yields a higher percentage of polysaccharides in comparisonto known media employed for producing meningococcal meningitis vaccine.

It is yet another object of the present invention to invent acomposition of a medium that yields a lower percentage of cellularbiomass in comparison with known media employed for producingmeningococcal meningitis vaccine.

It is yet another object of the invention to identify an optimummolecular size of N. meningitidis polysaccharides of serogroups A, C, Yand W-135 that confers broad spectrum immunogenic protection againstmeningitis.

It is yet another object of the present invention to invent an improvedcapsular polysaccharide diphtheria conjugate vaccine for meningococcalmeningitidis that prevent meningitidis deaths even in toddlers and be avaccine for all ages.

Another object of the present invention is to develop a process for theproduction of an improved capsular polysaccharide diphtheria conjugatevaccine and its use especially as vaccinal agent.

The invention which was the subject matter of U.S. Pat. No. 7,491,517was invented by this applicant and was well received and patented.However the need to reduce the costs in the medium preparation remains acontinuous one as this can bring down the costs of the vaccine making itaccessible to greater number of people.

BRIEF SUMMARY OF THE INVENTION

Methods for producing a quadrivalent polysaccharide vaccine of serotypesA, C, Y and W-135 for meningococcal meningitidis by mechanical means:The methods employ modified Neisseria meningitidis fastidious mediumspecially formulated to be more economical and cheaper than the previousNMFM medium but with the same yield of capsular polysaccharides,cellular biomass and endotoxins. The crude polysaccharides are isolatedand purified by ultra-filtration and gently treated with a polycationiccompound that precipitates the polyanionic capsular polysaccharides tomaximize the yield of precipitated polysaccharides from liquid cultures.The polysaccharides are then mechanically depolymerized, preferably bysonication. The pure polysaccharides were found in human clinical trialsto be highly effective against meningitis caused by N. meningitidis A,C, Y and W-135 serogroups. In the most preferred embodiment the purepolysaccharides are conjugated to carrier proteins of diphtheria ortetanus toxoid to provide broad spectrum protection to humans of all agegroups.

The present invention is directed to a method of preparing modified NMFMmedium comprising sodium chloride (NaCl), potassium phosphate dibasic(K2HPO4), ammonium chloride (NH4Cl), potassium sulphate (K2SO4),magnesium chloride ((MgCl2.6H2O), calcium chloride (CaCl2.2H2O),glucose, L-glutamic Acid, L-arginine, glycine, L-serine, L-cysteine.HCl,Fe (III) citrate, yeast extract (30 k dia-filtered) and antifoamdissolved in DI water. The specific formulation used in the experimentsconducted is given below.

Composition of Modified Neisseria meningitidis Fastidious Medium (NMFM)for Serogroups A, C, Y and W-135: (Grams Per Liter)

g/L or Component mL/L DI Water (initial) 800 mL Sodium Chloride (NaCl)5.60 Potassium Phosphate Dibasic 4.10 (K2HPO4) Ammonium Chloride (NH4Cl)1.00 Potassium Sulphate (K2SO4) 0.90 Magnesium Chloride ((MgCl2•6H2O)0.35 Calcium Chloride (CaCl2•2H2O) 0.03 Glucose 10.00 L-Glutamic Acid3.80 L-Arginine 0.10 Glycine 0.20 L-Serine 0.50 L-Cysteine•HCl 0.10 Fe(III) Citrate 0.04 Yeast Extract (30k dia-filtered) 0.1% Antifoam 0.1 mL

Filter sterilized glucose and amino acids were added to the autoclavedcool medium, which improved production of polysaccharides by 25%. Thisprocess allows non-degradation of heat sensitive sugars and amino acidsand eliminates batch feeding during the fermentation process forpolysaccharide production. The above modified medium is speciallyformulated to make the medium more economical and cheaper than theprevious NMFM medium and to produce results similar to previous NMFMmedia like increase in the production of capsular polysaccharide anddecrease in the production of cellular biomass. One more special featureof the medium is that the pH is maintained from about 6.5 to 7.0 duringthe fermentation process without using buffers and pH probes. Here inthis invention the phenol extraction step of the purification processfound in the prior art is replaced by activated carbon filtration toavoid any phenol interaction during purification. The isolatedpolyanionic polysaccharides are then precipitated with a polycationiccompound. The precipitated polysaccharides are then subjected toultra-filtration for the isolation of pure polysaccharides. The isolatedpure polysaccharides are depolymerized by sonication. These lowmolecular weight polysaccharides proved very effective when comparedwith other inventions. The human trials for pure polysaccharides ofserotypes A, C, Y and W-135 of this invention indicated very mildadverse side effects, none of which were severe, and also proved to bevery effective for humans above the age of 13 years and may provideeffective protection against meningococcal meningitis for humans abovethe age of 5 years.

In another preferred embodiment the pure low molecular weightpolysaccharides were conjugated to carrier proteins of diphtheriatoxoids to produce quadrivalent meningococcal meningitis conjugatedvaccine for the serotypes A, C, Y and W-135. This conjugated vaccineproved effective for all ages. The vaccine proved to be non-toxic andimmunogenic in animal trials using neonatal mice and mice of 7-8 weeks.The use of mice models in animal trials show that the conjugatedquadrivalent polysaccharide vaccine A, C, Y and W-135 may also beeffective in risk age groups (children below 2 years) and can immunizeeffectively humans of all ages.

The present invention is directed to a method of deriving anoligosaccharide from Neisseria meningitidis (NM) serogroups A, C, Y andW-135 and linking it between antigenic activated Neisseria meningitidisserogroups A, C, Y and W-135 polysaccharide and diphtheria toxoid, aprocess for preparing it and its use especially as vaccinal agent.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF DRAWINGS

FIG. 1 Neisseria meningitidis serogroup A Polysaccharides (PS)production in NMFM Media versus bacterial growth in 250 liter Fermentorwith 100-liters working volume.

FIG. 2 Neisseria meningitidis serogroup C Polysaccharides (PS)production in NMFM Media versus bacterial growth in 250 liter Fermentorwith 100-liters working volume.

FIG. 3 Neisseria meningitidis serogroup Y Polysaccharides (PS)production in NMFM Media versus bacterial growth in 250 liter Fermentorwith 100-liters working volume.

FIG. 4 Neisseria meningitides serogroup W-135 Polysaccharides (PS)production in NMFM Media versus bacterial growth in 250 liter Fermentorwith 100-liters working volume.

DEFINITION

Modified NMFM indicates Modified Neisseria meningitidis FastidiousMedium.

DETAILED DESCRIPTION OF THE INVENTION

N. meningitidis serogroup A, C, Y and W-135 Polysaccharides comprise thevaccine against meningitis. The goal of this experiment was to compareour invented medium with two commonly used cultivation media forproduction of polysaccharides. Our NM Fastidious Medium (NMFM) wascompared with Watson-Scherp and Catlin Media. The comparative criteriawas based on the final polysaccharide concentrations and the yieldcoefficient cell/polysaccharide (Y.sub.P/X). The kinetic parameters: pH,substrate consumption and cell growth. Cultivation of meningococcalserotypes was carried out in a 100 L New Brunswick® bioreactor, underthe following conditions: 80 L of culture medium, temperature 35.degree.C., 6% CO.sub.2, air flow 5 L/min, agitation frequency 120 rpm andvessel pressure 6 psi, without dissolved oxygen or pH controls. Thecultivation runs were divided in three groups, with 3 repetitions each.The cultivations using NM Fastidious Medium (NMFM) presented the bestresults: average of four serotypes final polysaccharide concentration at12 hours in 80 Liters=45.25 mg/L and Y.sub.P/X=0.13, followed byWatson-Scherp medium with results of 27.00 mg/L and Y.sub.P/X=0.07 andCatlin medium results of 22.5 mg/L and Y.sub.P/X=0.05 a respectively.The principal advantage we claim here is in the use of the NMFM forbetter vaccine production or polysaccharide yield than Watson-Scherp andCatlin media.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Several synthetic media were discovered for large-scale production ofmeningococcal meningitidis polysaccharide. Polysaccharide production inbatch process of Neisseria meningitidis serogroup C comparing Frantz,modified Frantz and Catlin 6 cultivation media. None of these mediaeliminated the problems associated with longer duration of fermentationand limitations on endotoxin production. Greatest emphasis was placed onthe cost of media components, not on the time associated withfermentation or with the expenses for endotoxin removal or with the timeassociated with the production of meningococcal polysaccharide vaccine.Though NMFM medium is more expensive than other synthetic media,large-scale production of meningococcal meningitidis polysaccharideusing NMFM medium eliminates the longer fermentation process. A majoradvantage of NMFM medium is the elimination of endotoxin in thepolysaccharide purification process for a given time.

The main aim of this invention is to make the medium more economical andcheaper thereby cutting down the cost of the vaccine, making itavailable to the public at reasonably affordable prices.

The detailed method of preparation of modified NMFM in various culturevolumes is described in the following paragraphs:

Composition of Modified Neisseria meningitidis Fastidious Medium (NMFM)for Serogroups A, C, Y and W-135: (Grams Per Liter)

g/L or Component mL/L DI Water (initial) 800 mL Sodium Chloride (NaCl)5.60 Potassium Phosphate Dibasic (K2HPO4) 4.10 Ammonium Chloride (NH4Cl)1.00 Potassium Sulphate (K2SO4) 0.90 Magnesium Chloride ((MgCl2•6H2O)0.35 Calcium Chloride (CaCl2•2H2O) 0.03 Glucose 10.00 L-Glutamic Acid3.80 L-Arginine 0.10 Glycine 0.20 L-Serine 0.50 L-Cysteine•HCl 0.10 Fe(III) Citrate 0.04 Yeast Extract (30k dia-filtered) 0.1% Antifoam 0.2 mLMedia PreparationModified NMFM Media Preparation for ABI GC5000 250 L Fermentor (130 L)

To prepare medium for the ABI GC 5000 Fermentor (SOP 4020), the mixingtank is filled with an initial volume of DI water and connected to powersource. Using a digital balance (SOP 4034) and weighing boats, eachcomponent of the modified NMFM media is weighed for preparing thedesired volume. Ingredients are added to the mixing tank one by one asper the order listed in the table and each ingredient is allowed todissolve before the next one is added. Required amount of antifoam isadded once the other chemicals have been dissolved. DI water is added tothe marked volume on the view glass of the mixing tank (SOP 4039). Afterall ingredients have been dissolved using the medium tubing and theMasterflex Digital I/P pump (SOP 4022) the medium is transferred intothe fermentor. Medium tubing is connected to the bottom valve of themixing tank using a tri-clamp and the medium is transferred into thefermentor through the media transfer port. The medium should besterilized after the transfer is complete, (refer to SOP 4020 forsterilization procedure). Glucose solution (SOP 9024) is preparedseparately, autoclaved, and added after the remaining medium componentsare sterilized. In a biological safety cabinet, 33.3 ml 33.3% glucosesolution/liter of medium is poured into a large sterile aspirator bottleassembly. The solution is pumped into the fermentor using the MasterflexDigital I/P pump (SOP 4022).

Media Preparation for 30 L Chemap Fermentor (20 L)

To prepare medium for the Chemap Fermentor (SOP 4019), a 4 L beaker isfilled with about 3.5 L of DI water and a stir bar is kept inside thebeaker. The beaker is placed on a stir plate for stirring (SOP 4006).Using a digital balance and weigh boats, each component of the modifiedNMFM media is weighed for preparing the desired volume. The actualamount weighed for each ingredient is marked on the modified NMFM mediumdata form. Ingredients are added to the beaker one by one as per theorder listed in the table and each ingredient is allowed to dissolvebefore the next one is added. Required amount of antifoam is added tothe medium and the ingredients are allowed to mix thoroughly until itdissolves. The magnet is removed using a stir bar retriever. 4 L ofmedium is poured into the Chemap Fermentor using a funnel and the DIwater is adjusted to the desired level. The medium is sterilizedaccording to SOP 4019. Glucose solution (SOP 9024) is preparedseparately, autoclaved, and added after the remaining medium componentsare sterilized. In a biological safety cabinet, 33.3 ml 33.3% glucosesolution/liter of medium is poured into a small sterile aspirator bottleassembly and the solution is pumped into the fermentor using theMasterflex Digital I/P pump (SOP 4022).

Media Preparation for Neisserial Shake Flask Fermentation:

The medium has four parts of component making.

Part A: Making yeast extract supplement

Part B: Making 5× nutrient mix

Part C: Making 100× Calcium and magnesium salt mix

Part D: Making Glucose supplement

Part A: Making of Yeast Extract Supplement (1% Y.E Supplement):

11 g of yeast extract is weighed and dissolved in 1100 ml of DI water bymagnetic stirring. The obtained yeast extract solution is passed through0.2 μm Nalgene filter. The filtrate is passed through VIVA flow 30Kdiafiltration unit. The retentate is recycled and the filtrate iscollected to remove large molecular weight glucans. Diafiltration isstopped once 1 L filtrate is collected. Residual retentate (about 100ml) is discarded.

Part B: Making of 5× Nutrient Mix:

Ingredient Grams/L Sodium Chloride (NaCl) 28.0 Potassium PhosphateDibasic 20.50 (K2HPO4) Ammonium Chloride (NH4Cl) 5.00 Potassium Sulphate(K2SO4) 4.50 L-Glutamic Acid 19.0 L-Arginine 0.50 Glycine 1.0 L-Serine2.50 L-Cysteine•HCl 0.50 Fe(III) Citrate 0.2

The above ingredients are weighed and dissolved in 1 L DI water toconstitute 5× nutrient mix.

Part C: Making of 100× Calcium and Magnesium Salt Mix:

Ingredient Grams/L Magnesium Chloride ((MgCl2•6H2O) 35.0 CalciumChloride (CaCl2•2H2O) 3.0

The above 100× calcium and magnesium salt mix is prepared in DI waterand it is filter sterilized using 0.2 micron Nalgene filter.

Part D: Making of Glucose Supplement:

330 grams of glucose supplement is weighed per liter of Di Water (33%)and filter sterilized using 0.2 micron Nalgene filter.

Preparation of 1 L JN NMFM Medium Using Above Four Parts:

200 ml of part B is taken in a clean measuring jar and 100 ml of part Ais added to it. The volume is made up to 965 ml with DI water,transferred to an Erlenmeyer flask and autoclaved at 121° C. for 15minutes. 1 ml of part C and 33.3 ml of part D are added asepticallyafter the autoclaved medium attains room temperature.

The aim of the study was to describe the dynamic behavior of thebioprocess system of Neisseria meningitidis that can be used in futurefor the control and optimization of the industrial process of capsularpolysaccharide production. Inoculation procedure and cultivationconditions were as described later in the document. Samples ofcultivation were collected at pre-established time intervals andanalytical assays were conducted to obtain the rate of microbial growth,glucose uptake, and polysaccharide production. An analysis of thekinetics of capsular polysaccharide production by Neisseria meningitidisserogroups was conducted. Based on microorganism behavior and transientcharacteristics (common to processes operated in batch operation mode)such as variations in glucose concentration, accumulation of metabolicproducts and availability of dissolved oxygen, a standard set ofbioprocess conditions were developed.

Microbial Growth:

The specific microbial growth rate was directly proportional to theconcentration of dissolved oxygen in the cultivation medium. Microbialgrowth was limited by concentration of glucose and no growth substrateinhibition effects were observed in the glucose concentration studies.Glucose consumption was affected by the availability of dissolvedoxygen. During the logarithmic phase, the bacterial growth in the mediumshowed high oxygen concentration and low glucose uptake, and this wasmuch lower than the highest specific microbial growth rates. Glucoseuptake increased when an oxygen concentration of zero was achieved. Thusthe rate of glucose (carbohydrate) metabolism by Neisseria meningitidiswas determined by the availability of oxygen. A buildup of capsularpolysaccharide formation occurred because the limited availability ofoxygen favored the specific polysaccharide production of serotypes A, C,Y, and W-135. The existence of a maximum quantity of surfacepolysaccharide for each serotype was observed under the conditionsdescribed above.

The process of capsular polysaccharide production by Neisseriameningitis serogroups was performed in a 100 Liter bioreactor.Experimental results showed that the availability of dissolved oxygen inthe cultivation medium determined kinetics of the N. meningitidisbacterium. Higher concentration of oxygen favored microbial growth anddecreased the specific capsular polysaccharide production. This could berelated to the use of a common lipid intermediate, either in theconstruction of cell walls, an essential structure for bacterialsurvival, or in the biosynthesis of capsular polysaccharide. The morerapid accumulation of capsules under low concentration of dissolvedoxygen could also be associated with the need to produce this cellprotection structure in situations of stress, such as limitedavailability of oxygen.

The presence of Class 4 proteins of Neisseria meningitidis is known tobe anti-bactericidal. Therefore we used Neisseria meningitidisserogroups A, C, Y and W-135 vaccine strains, which were deleted forClass 4 proteins, in vaccine production by the purification ofpolysaccharides using novel methodology to produce toxin-free vaccine.

Immunogenicity, pyrogenicity, and toxicity of purified polysaccharideswere determined in animals and humans. Immunogenicity of vaccinecandidates were tested by ELISA and Serum Bactericidal Assays (SBA)using animal and human sera which showed high serum titers in SBA andhigh reactivity titers in ELISA in vitro experiments.

Production of Low Molecular Weight Polysaccharides for MeningococcalMeningitis Vaccine—Preparation and Formulation

Natural meningococcal meningitidis polysaccharide is about 500,000 to1,500,000 daltons. A novel extracellular low-molecular-weightpolysaccharide was detected within extracellular class 4 deleted mutantsof Neisseria meningitis serotype cultures.

The present invention is directed towards a non-chemical method(sonication) to make low molecular weight polysaccharides and to produceconjugated meningococcal meningitidis polysaccharides with minimum rangeof 5100 to 9900 Daltons size.

Compositional analysis, methylation analysis, and nuclear magneticresonance analysis revealed that this low-molecular-weightpolysaccharide was composed of the same polysaccharide repeating unitpreviously described for the high-molecular-weight form of thepolysaccharides synthesized from Neisseria meningitis serotypes.

The purified polysaccharides contain high and low molecular weightExtracellular polysaccharides (EPS). Magnetic sonication was done at4.degree. C. for 2 hours to obtain soluble low molecular weight EPS. Thesoluble low molecular weight EPS were collected and analyzed by Massspectrometry which indicated that its size was consistent with a dimericform of the polysaccharide repeating unit. High-molecular-weight EPSwere then removed from concentrated supernatants by centrifugation(12,000.times.g for 10 min). Low-molecular-weight, ethanol-solublepolysaccharides were then purified from concentrated supernatants usinggel permeation chromatography.

EXAMPLE Fermentation Procedure

The working Seed Bank stocks of Neisseria meningitidis A, C, Y, W-135were kept frozen in glycerol solution at −80.degree. C. These stocktubes were thawed in running cold water and inoculated in Columbia agarplates after the outer surface of the tube was disinfected with ethanol.The plates were incubated overnight (18 hours) at 37.degree. C. in anincubator with 6% CO.sub.2 atmosphere. The cultures were re-streaked onfresh plates to isolate pure cultures of Neisseria meningitidis andincubated at 37.degree. C. in 6% CO.sub.2 atmosphere overnight for 12hours. Bacterial colonies from two plates were collected with a sterilecotton swab and suspended in two 10 ml aliquots of ShedularsBroth®(Remel, Inc.®) in separate 15 ml centrifuge tubes and re-suspendedinto 50 ml media contained in a 200 ml flask. The culture was allowed togrow in a shaker at 125 rpm, 35.degree. C. and normal atmosphericpressure for about 3 hours and then transferred into a 1 Liter flaskcontaining 200 ml pre-warmed medium and again incubated at 125 rpm,36.degree. C. for 12 hours to form a seed culture. An absorbance of 1.5at 600 nm is considered equivalent to 500 Klett units. The culture wasthen transferred to a 2 Liter conical flask, containing 500 ml of thesame medium, inoculated with 50 ml of the inoculum and incubated underthe conditions previously described. Inoculum from eight flasks (theratio of inoculum to the media is 1:10 or 8 L:80 L) were used toinoculate the bioreactor (New Brunswick® model MPP 80-total capacity 100L) with 80 L of medium. The cultivation conditions were: temperature35.0.degree. C.; air flow rate 5 L/min (0.125 vvm, superficialaeration); agitation frequency 120 rpm (with 2 Rushton six blade discturbines); vessel head space pressure 6 psi; height and diameter of thevessel 72 and 40 cm, respectively; turbine diameter 16.5 cm, one locatedat 10 cm from the vessel bottom and the other at 35 cm. Four baffleswere installed to enhance the mixture efficiency. The oxygen volumetrictransfer coefficient (k.sub.La) was near 0.07 min.sup.-1 before theinoculation (t=0 h). The batch cultivations run under the sameoperational conditions, were divided into three groups, each one withthree repetitions: the first one with Watson-Scherp medium, the secondwith Catlin medium; and the third with modified NMFM medium.

Analysis:

Cell concentration was articulated as dry biomass, as determined bycentrifugation of a sample at 10,000.times.g, followed by drying thepellet at 60.degree. C. for 48 hrs. Polysaccharide concentration wasassessed after bacterial cell removal and was precipitated by addingCetavlon™ to the sample. The supernatant was removed aftercentrifugation and the precipitated biomass was re-suspended in a oneMolar CaCl.sub.2.2H.sub.2O solution to obtain supernatant forpolysaccharide determination using the following method: a samplecontaining 10-70 g of sialic acid is taken in a 16.times.150 mm glasstube with the sample volume brought up to 500 g/L. Standard solutions ofsialic acid were prepared using 20, 40, 60, 80, and 100.mu.g, and eachwas made up to 500 g/L to which was added 50 micro-liters resorcinolreagent. The tubes were placed in boiling water bath for 15 min. If thetube shows blue/purple/brown color, it indicates that the samplecontains sialic acid. Absence of blue color indicates that sialic acidis not present. A dark brown color indicates that the sample has eithertoo high a concentration of sialic acid or that it is not pure enough.The tubes were cooled to room temperature (20-25.degree. C.) in coldwater bath and 1 mL (2.times. sample volume) of extraction organicsolvent was added into each test tube. The tubes were shaken vigorouslyand left at room temperature until the organic solvent layer separatescompletely from the aqueous phase. The top organic phase was transferredto a cuvette and the absorbance was determined against pure organicsolvent in a spectrophotometer at 580 nm. Absorbance was compared with astandard curve for quantification, which detects the polysaccharidemonomers (sialic acids) formed after acid hydrolysis. Yield coefficientwas calculated by the ratio between polysaccharide production and cellbiomass generated (Y.sub.P/X) for a given cultivation time. FIGS. 1-4show the production associated with polysaccharides in the bacterialpopulation up to the 20.sup.th hour of incubation.

The compositions of the culture media are:

Modified Neisseria Meningitidis Fastidious Medium (NMFM) for serogroupsC, Y and W-135: (grams/Liter): Sodium chloride (NaCl), potassiumphosphate dibasic (K2HPO4), ammonium chloride (NH4Cl), potassiumsulphate (K2SO4), magnesium chloride ((MgCl2.6H2O), calcium chloride(CaCl2.2H2O), glucose, L-glutamic Acid, L-arginine, glycine, L-serine,L-cysteine.HCl, Fe (III) citrate, yeast extract (30 k dia-filtered) andantifoam dissolved in DI water.

Watson-Scherp Medium: grams/Liter: Sodium phosphate, dibasic 2.500; Soypeptone 5-30; Monosodium Glutamate 5.000; Potassium Chloride 0.103;Magnesium sulfate 0.732; L-Cysteine 0.016; Glucose 11.250

Catlin Medium (MCDA) Catlin, (in mM: NaCl, 100; KCl, 2.5; NH.sub.4Cl,7.5; Na.sub.2HPO.sub.4, 7.5; KH.sub.2PO.sub.4, 1.25; Na3C6.H5.O7.2H2O,2.2; MgSO.sub.4.7H.sub.2O, 2.5; MnSO.sub.4.H.sub.2O, 0.0075; L-glutamicacid, 8.0; L-arginine.HCl, 0.5; glycine, 2.0; L-serine, 0.2; L-cysteineHCl.H.sub.2O, 0.06; sodium lactate, 6.25 mg of 60% syrup/mL of medium;glycerin, 0.5% (v/v); washed purified agar, 1% (wt/vol)CaCl.sub.2.2H.sub.20, 0.25; Fe.sub.2(SO.sub.4).sub.3, 0.01)

Kinetics:

Kinetics of glucose consumption verses pH was evaluated for the variousmedia. When the Watson-Scherp medium was used, 6 g/L of glucoseconsumption was observed at the end of the cultivation; with modifiedNMFM medium the residual concentration of the substrate was 3 g/L andwith Catlin medium glucose consumption was between 5-6 g/L. Theconsumption of glucose during cultivation yielded acid metabolites inWatson-Scherp medium and Catlin medium. These results indicate thatthese media require adjustment of pH during the fermentation process.Modified NMFM medium does not require adjustment of the pH throughoutcultivation of polysaccharide or vaccine production and provides minimalstress on the bacteria during the fermentation process. This factindicating the absence of acid metabolites, also indicates thatsequential consumption of amino acids (as a source of carbon) may havetaken place.

The association between polysaccharide production and biomass isextremely important in endotoxin-free large-scale production. Duringcultivation of N. meningitidis serogroups A, C, Y, W-135 in a bioreactorit is crucial to pay attention to two criteria: attaining the maximumpolysaccharide concentration at the end of the cultivation in thebioreactor (P.sub.f) and simultaneously attaining the minimum celldebris (biomass) yield factor (Y.sub.P/X) which is important forpolysaccharide purification. The cell debris is nothing but endotoxincontaminant that must be removed in the purification process.

Statistical Analysis:

Statistical analysis was performed using test “t” at the 5% significancelevel to compare the data obtained from the three media used in thisstudy. Greater final concentrations of polysaccharide (P) and greatercell/polysaccharide yield factors (Y.sub.P/X) were obtained from groupexperiments 1 to 3, where the modified NMFM medium used resulted in anaverage of 45.25 mg/L. In addition, statistical tests on the biomassvalues determined at the end of the cultivations (X.sub.max) showed thatthe use of Watson-Scherp medium resulted in production of a largebiomass of N. meningitidis and did not give best values for the yieldfactor (Y.sub.P/X), compared to experiments carried out using themodified NMFM medium. This implies that there was a higher concentrationof dry cellular biomass production when using Watson-Scherp medium andthe lowest was found when using modified NMFM medium.

The results obtained from the experiments that used the modified NMFMmedium with a glucose concentration of 10.0 g/L, showed that theresidual glucose value at the end of the cultivation was lower than thatobtained in Watson-Scherp medium and Catlin medium. Kinetics of nitrogenconsumption by N. meningitidis during polysaccharide production usingthe modified NMFM medium showed that adding the nitrogen source, in thepresence of excess glucose, resulted in a greater production ofpolysaccharides. In 12 hours, the polysaccharide production usingmodified NMFM medium showed 45.25 mg/L at neutral pH, minimal dry massof 0.32 g/L with lower utilization of carbon source compared toWatson-Scherp and Catlin media. The advantages of the modified NMFMmedium are lower costs and easier cultivation and purification stages inthe polysaccharide production process.

Procedure for the Production of Capsular Polysaccharide

Capsular polysaccharide production by Neisseria meningitidis serogroupsA, C, Y, and W-135 was studied in batch experimental runs. Theexperiments were conducted in a set of 100 L bioreactors with 80% ofmodified NMFM cultivation medium. Cultivation temperature and pH werecontrolled at optimal pre-established values. The dynamic behavior ofthe bacteria was analyzed based on biomass growth, glucose uptake,polysaccharide production, and dissolved oxygen time profile obtained ina set of experimental runs with initial concentrations of glucose thatvaried from 5 to 13.5 g/L.

The preset set of controlled conditions for the production ofpolysaccharides maximized the accumulation of polysaccharides, lowbiomass, and endotoxin accumulation due to the lack of new bacterialcell formation. Although glucose was completely consumed, there was nosignificant difference in the final concentration of polysaccharidesbetween the bioreactor runs using individual serotypes of N.meningitidis. Final concentrations of biomass were very similar amongall serotypes for all experimental runs. The medium formulation ofmodified NMFM has limited phosphate availability, and resulted in lowerbiomass production, glucose consumption, endotoxin concentration,dissolved oxygen, better pH balance, and greater polysaccharideproduction for all Neisseria meningitidis serotypes. Thus, the designedpreset conditions shall be employed for implementation of future processcontrol and optimization of the industrial polysaccharide vaccineproduction with modified NMFM medium for Meningococcal meningitisserotypes A, C, Y, and W-135.

Concept for Neisseria Meningitidis Medium Invention:

Modified NMFM medium is more economical and cheaper than the previouslyinvented NMFM medium (U.S. Pat. No. 7,491,517) which recites the samecharacteristics as follows:

NMFM medium is a highly-enriched bacteriological medium useful forgrowing fastidious bacteria. The bacterial cell growth in this medium isfaster than in any other known synthetic and non-synthetic media. NMFMis useful for production of high quantities of toxin-free polysaccharidein a duration of less than or equal to 12 hours. Filter sterilizedglucose and amino acids were added to the autoclaved cool medium, whichimproved production of polysaccharides by 25%. This type of processallowed non-degradation of heat sensitive sugars and amino acids andeliminated batch feeding during the fermentation process forpolysaccharide production. Use of NMFM medium for Meningococcalmeningitis vaccines saves almost 50% cut-off time in the fermentationprocess and purification of toxins, and results in clinically-provensafer vaccine production as compared to the use of Watson-Scherp andCatlin media, which require longer periods of fermentation and a moreintensive toxin purification process. We used calcium carbonate(CaCO.sub.3) to balance the pH of the medium, as opposed to the use ofcalcium chloride (CaCl.sub.2), which can make media more alkaline duringfermentation. Ionized calcium is the key buffer that helps to maintainthe acid/alkaline balance in NMFM medium. We allowed the mutant strainsof Neisseria meningitidis serotypes to grow slowly in a short period ofincubation to reach lower maximal optical density and to produce moreendotoxin-free polysaccharides (PS) than the use of standard media. Coxet. al., reported that the NMB1638 gene of Neisseria meningitidis wasresponsible for a lipopolysaccharide (LPS) containing lipid A that wascharacteristically phosphorylated with multiple phosphate andphosphoethanolamine residues. Mass spectroscopic analyses of the LPS ofNeisseria meningitidis strains that had been inactivated by a specificmutation indicated that there were no phosphoethanolamine residues.Neisseria meningitidis produces two types of toxins called exotoxins andendotoxins. Exotoxins are released from bacterial cells and may act attissue sites removed from the site of bacterial growth. Endotoxins arecell-associated substances that are structural components of the cellwalls. However, endotoxins are released from growing bacterial cells orfrom cells which are lysed as a result of effective host defense. Hence,bacterial toxins, both soluble and cell-associated, may be transportedby blood and lymph and cause adverse reactions in humans.Lipopolysaccharides are considered the major endotoxin in polysaccharideproduction. Removal of or minimal supplementation of organic phosphatesin liquid cultures is very important in meningococcal polysaccharideproduction to reduce the production of endotoxins.

Gotschlich et. al, first reported effective method for purification ofmeningococcal polysaccharides from liquid cultures. Cationic reagentCetavlon™ (hexadecyltrimethyl ammonium bromide) was used to precipitateanionic polysaccharides.

Inorganic phosphates (P.sub.i) are required for any bacterium tofunction as constituents of nucleic acids, nucleotides, phospholipids,lipopolysaccharides (LPS) or toxins, and teichoic acids. Inphosphate-deficient NMFM medium, the bacterium utilizes itsintracellular phosphate reserve for its cellular function at minimumrates for production and release of undesirable LPS, or toxins, into themedium at minimal level. The NMFM medium does contain minimal inorganicphosphate (P.sub.i) salts, but is buffered by 10 mMmorpholinepropanesulfonic acid (MOPS; pH 7.0). Due to the stress inducedby pH balance combined with (P.sub.i) deficiency of the medium, NMFMmedium allowed mutant strains of Neisseria meningitidis serotypes togrow more slowly, reach lower maximal optical densities, produce lesstoxins, and produce more polysaccharides (PS) than the standard media.Neisseria meningitidis serotypes synthesize capsular polysaccharidesthat are used as vaccine candidates. These molecules are produced bybacterium as a capsule under strong stressful conditions (bothnutritional and physiological stress as stated above) and are tightlyassociated with the cell assembled as capsular polysaccharides (CPS)which surround the cell surface. When they are liberated into the mediumthey are called extracellular polysaccharides (EPS).

The capsule expressed by N. meningitidis is categorized as a group IIcapsule based on the similar chemical and physical properties ofcapsular polymers. Serogroup A is composed of (.alpha.1.fwdarw.6)-linkedN-acetylmannosamine-1-phosphate. The capsules expressed by each of theother major invasive meningococcal serogroups Y and W-135 are composedof alternating units of D-glucose and D-galactose and sialic acid,respectively. The capsular polysaccharides of serogroups C are composedentirely of sialic acid in an (.alpha.2.fwdarw.8) or an(.alpha.2.fwdarw.9) linkage.

Phosphatase activity (P.sub.i) and pH balance induced increasedpolysaccharide production by Neisseria meningitidis isolates. Data arethe average of the means of at least three independent experiments inwhich each experimental mean was derived from PS extracts of threeseparate cultures. Cells were incubated for 12 h in NMFM with (+) orwithout (−) phosphorus.

The tables below indicate the phosphatase activity of the respectiveserogroups shown below. (+) plus phosphates (+P.sub.i) in the tablesbelow indicate, Thiamine pyrophosphate 0.10 g; K.sub.2HPO.sub.4 4.00 g,Na.sub.2HPO.sub.4 7.5. (−) minus phosphates (−P.sub.i) in the tablesbelow indicate, Morpholinepropanesulfonicacid (MOPS; pH 7.0).

TABLE 1 Serogroup A PHOSPHATASE Culture Assay ACTIVITY Phosphate pHmg-polysaccharides +plus −minus 5.0 7.0 0 hours 0 0 0 0 3 hours 8 7.20.92 7.2 8 hours 17 35 0.8 35 12 hours 20 43 0.4 43

TABLE 2 Serogroup C PHOSPHATASE Culture Assay ACTIVITY Phosphate pHmg-polysaccharides +plus −minus 5.0 7.0 0 hours 0 0 0 0 3 hours 3 9 0.859 8 hours 15 39 0.8 39 12 hours 21 48 0.4 48

TABLE 3 Serogroup Y PHOSPHATASE Culture Assay ACTIVITY Phosphate pHmg-polysaccharides +plus − minus 5.0 7.0 0 hours 0 0 0 0 3 hours 2 8 0.58 8 hours 3.7 25 0.2 25 12 hours 8 43 0.13 43

TABLE 4 Serogroup W-135 PHOSPHATASE Culture Assay ACTIVITY Phosphate pHmg-polysaccharides +plus −minus 5.0 7.0 0 hours 0 0 0 0 3 hours 1.7 7.20.5 7.2 8 hours 2.4 41 0.18 41 12 hours 6 47 0.1 47Preparation of Meningococcal Meningitis Polysaccharide Vaccine:

Proteins and nucleic acid contaminants were precipitated with ethanolfollowed by polysaccharide precipitation with Cetavlon™, a polycationiccompound used specifically to collect polyanionic polysaccharides. Theresidual contaminants were further removed by proteinase digestion andultra-filtration. In this invention, we also used the polycationiccompounds to specifically collect polyanionic polysaccharides afterprecipitating with Cetavlon™ which gave high purity polysaccharidevaccine components. Overnight, CaCl.sub.2 was retained with Cetavlon™precipitated polysaccharide at 4.degree. C. The polysaccharides werefurther precipitated by slow addition of ethanol to collectpolysaccharide residues and to remove contaminants in the preparation togive absolute purification of the vaccine compound. The phenolextraction step as described in the prior art was replaced withactivated carbon filtration. Activated carbon and Sephacryl gelfiltration yielded high purity and quantity of polysaccharide vaccinecomponents.

Polysaccharide Production in Modified NMFM Medium

The Neisseria meningitidis serotypes were grown in separate 100-Lbioreactors in modified NMFM medium for eighteen to twenty hours (asdescribed earlier). Absorbance unit: Optical Density (OD) of bacterialgrowth of 10 at 600 nm, after a fermentation process of 12 hours, waschosen for the cultivation of polysaccharides from N. meningitidis.Formaldehyde (36.5-38%) 1% (v/v) was added to the bioreactors at 25 psito kill the bacteria and then centrifuged (5,000.times.g for 30 min) toremove bacterial cells. The supernatant was collected, treated with 100%ethanol by slow addition with agitation and centrifuged to collect theprecipitate. The precipitate was re-dissolved in water andre-precipitated three times with ethanol by slowly adding 80% (v/v)ethanol, followed by centrifugation. The crude polymers werefractionated by stepwise precipitation with 1%hexa-decyl-tri-methyl-ammoniumbromide (Cetavlon™) at pH 7.0. at4.degree. C. overnight.

The precipitate was collected by centrifugation and re-suspended inwater and 10% Cetavlon to a final concentration of 0.1% (w/v) and anequal amount of 0.9 molar CaCl.sub.2 was then added to make a finalconcentration of 1 mM and the solution left overnight with continuousmixing or agitation at 4.degree. C. to remove endotoxin. The supernatantwas collected by centrifuging at 9000 rpm. Cold ethanol was added to thesupernatant to a final concentration of 25% and allowed to stand at4.degree. C. for 2 hours. The supernatant was collected by centrifugingat 5000 rpm for 40 min. Low molecular mass residual contaminants wereremoved with proteinase K digestion and filtered through activatedcarbon to remove trace organic compounds, repeatedly until OD.sub.275 nmwas <0.1. CPS was further purified by using the Sephacryl 200 gelfiltration column using 50 mM ammonium formate elutions.

Meningococcal Oligosaccharide Linked Polysaccharides and DiphtheriaProtein Conjugate Vaccine (PSOSDT)

In the preferred embodiment a method was developed for couplingcarboxylic acid-containing oligosaccharides linked to activatedpolysaccharides and diphtheria protein for vaccine preparation.Oligosaccharides of Neisseria meningitidis serogroups A were isolatedand the carboxylic acid at 2-keto-3-deoxyoctulosonic acid of the OS waslinked through adipic acid dihydrazide to pre-activated polysaccharidesand carrier protein as per Che-Hung Lee 2009. Where the carrier proteinused in the present invention is diphtheria toxoid (DT).

The antigenicity of the OS linked diphtheria conjugated topolysaccharides was measured by enzyme-linked immunosorbent Assay(ELISA) and Serum Bactericidal Assay (SBA) using meningococcalvaccinated human antibodies. The OS-polysaccharide-protein conjugatesderived from meningococcal vaccine may therefore be vaccine candidatesto prevent meningitis caused by meningococci effectively, may be usedfor all ages and longer immunity against meningococcal infections causedby the serogroups A, C, Y and W-135 proved by human clinical trial.

Production of Polysaccharide-Oligosaccharide-Diphtheria Toxoid ConjugateVaccine (PSOSDT)

The following method was used to link meningococcal oligosaccharides topolysaccharides and diphtheria protein to produce a conjugate vaccinefor all ages.

The Production of Polysaccharide-Oligosaccharide-Diphtheria ToxoidConjugate Vaccine (PSOSDT) has Five Major Steps:

Step 1: Isolation and purification of capsular polysaccharides from fourmeningococcal serogroups A, C, W-135 and Y

Step 2: Derivatization of oligosaccharides (OS)

(A) Isolation of lipo-oligosaccharide from four meningococcal serogroupsA, C, W-135 and Y

(B) Removal of lipid A from Lipo-oligosaccharide to obtainoligosaccharides:

Step 3: Purification of Diphtheria toxoid (DT)

(A) Activation of Diphtheria toxoid (DT)

Step 4: All the three components PS, OS and DT were combined in adefined ratio for conjugation

Step 5: Final formulation

Step 1: Isolation and Purification of Capsular Polysaccharides from FourMeningococcal Serogroups A, C, W-135 and Y

Isolation of polysaccharides from four meningococcal serogroups is doneusing culture supernatant-CTAB precipitation. Seed cultures wereinitially streaked on chocolate agar plate and incubated at 37° C.overnight in the presence of 5% CO₂. Grown culture is tested for itspurity by gram staining, oxidase test and serogroup specificagglutination. Confirmed culture is streaked again on another chocolateagar plate for one more passage. Overnight healthy growth is transferredto 20 ml liquid broth and grown in a shaker incubator. 20 ml activegrowth is transferred to 180 ml broth in a 1 L flask. Active exponentialgrowth of 200 ml culture is inoculated in 1.8 L broth in 4 L Erlenmeyershake flasks. After reaching stationary state, culture is continued toincubate further 3-4 h. Later the shake flask culture is inactivated at56° C. for 45 minutes. After bringing back to room temperature culturepellet is separated by centrifugation, and supernatant was given 0.1%CTAB and kept at 4° C. overnight. Crude polysaccharide precipitate isthen separated by centrifugation. It is suspended in water and mixedwith equal volume of 2M CaCl₂ to adjust to a final concentration of 1MCaCl₂ and allowed to mix for 2 h at room temperature. To this suspensionwas given 25% final concentration of ethanol to precipitate nucleicacids and proteins. Pellet is again separated by centrifugation.Supernatant was given final concentration of ethanol to 80%, mixed welland allowed to precipitate overnight at 4° C. Precipitatedpolysaccharide is further purified by nuclease enzyme followed byprotease enzyme and dialyzed to remove nucleotides and peptides.Supernatant was given 0.1M CaCl₂ and subjected to ultracentrifugation toremove LOS. Ultra-supernatant was then dialyzed and concentrated todryness. The polysaccharide is further purified by size exclusion and/orhydrophobic interaction column chromatographic methods. Purifiedpolysaccharide is tested for any levels of contaminating nucleic acids,protein or endotoxin. Obtained vaccine grade polysaccharide structureswere verified by ¹H NMR.

Step 2: Derivatization of Oligosaccharides (OS)

ADH (Aldrich Chemical Co., Milwaukee, Wis.) was coupled tooligosaccharides by carbodiimide-mediated condensation with1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) andN-hydroxysulfosuccinimide (sulfo-NHS) (Pierce) as per (51)

Isolation of Lipo-Oligosaccharide from Four Meningococcal Serogroups A,C, W-135 and Y:

Lipo-oligosaccharide from individual serogroup is isolated from culturepellet with hot phenol-water method. Water layer and phenolic layerafter extraction were separately processed for LOS yields. Both layerswere extensively dialyzed against tap water and later against DI water.Dialyzed material was concentrated by lyophilization to dryness.Concentrated material was run by sephadex G-150 size exclusion columnand fractions were analyzed for neutral sugar. Sugar positive materialwas pooled and concentrated according to expected sizes. Later sampleswere run DOC-PAGE gels to stain the LOS by periodate-silver nitratestaining method. LOS was transferred to PVDF membrane and western wererun to see the identity of individual serogroups.

Removal of Lipid a from Lipo-Oligosaccharide to Obtain Oligosaccharides:

Purified LOS is treated with 1% acetic acid at 90° C. for 2 h forbreaking the ketosidic linkage between oligosaccharide and lipid A.After the hydrolysis the mixture is extracted with chloroform:methanol(2:1) to separate lipid A into organic layer. Aqueous layer is dialyzedand concentrated. Sugar content is estimated. Oligosaccharide isestimated by NMR and sugar analysis to confirm the serogroup specificsaccharide.

Step 3: Purification of Diphtheria Toxoid (DT)

Diphtheria toxoid was concentrated and passed through a Sephacryl S-300column equilibrated with 0.9% NaCl containing 0.02% sodium azide. Theprotein concentration was measured by the micro-bicinchoninic acidmethod (Pierce, Rockford, III.) (50), and bovine serum albumin (BSA) wasused as a standard.

Activation of Diphtheria Toxoid (DT):

Activation of DT to contain hydrazide groups (DT, 4.0 mg/mL) wasactivated with 0.40M hydrazine in the presence of 20 mM EDC, 0.1M2-morpholinoethanesulfonic acid (MES), pH 6.5 at 20-24° C. (Lee et al).The protein concentration of the resulting DT-hydrazide (DTH) sample wasdetermined by Lowry assay or BCA assay using BSA as reference. Thehydrazide content was determined by TNBS assay using ADH as reference.The degree of activation (DA, number of hydrazide per DT molecule) wascalculated from the molar concentrations of hydrazide and DT assuming150,000 kDa for the molecular weight of DT which is similar resultspublished by Lee et. Al.

Step 4: all the Three Components PS, OS and DT were Combined in aDefined Ratio for Conjugation

Obtained purified PS and OS were considered at predetermined ratios.They are separately activated with sodium periodate. PS was size reducedto suitable levels before considered for activation by 0.1% acidhydrolysis in case of A, W135 and Y. Group C is not considered for sizereduction as C polysaccharides can be size reduced during periodateactivation.

Diphtheria toxoid is activated by hydrazine to create hydrazide groupson the protein. Serogroup specific activated PS and activated OS arecombined at a predetermined ratio with activated DT to conjugate. Afterestablishing covalent linkage, unused active aldehyde groups were cappedusing sodium borohydride reduction. Unconjugated DT, PS and OS areremoved by size exclusion chromatography. Finally in this conjugation weend-up creating three populations a) PS-DT-OS b) PS-DT c) OS-DT at4:3:0.5 ratio.

Step 5: Final Formulation

The above procedure arrived at an aseptically formulated concentrationwhere the vaccine formulation will finally have doses in the ratio of8:8:1 (PS:DT:OS) based on polysaccharide and DT contents. For example,each dose contained: (A) 4 ug PS:4 ug DT:0.5 ug OS, (B) 2 ug PS:2ugDT:0.25 ug OS.

Results

Animal Pharmacology and Toxicology Studies

Animal studies conducted at Spring Valley Laboratories, Woodbine, Md.,USA, under the guidance from JN International, Inc, USA involving 24Balb/c mice and 24 Neonatal mice have demonstrated that NmVac A, C, Y &W-135 DT conjugate is safe and non-toxic. In-vitro bactericidal assaysconducted at Central Research Institute, Nebraska has demonstrated thatNmVac A, C, Y & W-135 DT conjugate elicited good immune responseproviding sero-conversion rates as measured by bactericidal antibodyassays: Sensitivity: Group A: 81%, Group C: 87%, Group Y: 90% and GroupW-135: 82%; Specificity: Group A: 86%, Group C: 82%, Group Y: 91% andGroup W-135: 93%.

It can be easily understood by persons of ordinary skill in the art thatthe modified NMFM Medium (Neisseria Meningitidis Fastidious Medium) canhave several other possible combinations of the ingredients of themedium and the embodiment of the modified NMFM medium described hereinis limited only by the claims made herein.

Human Clinical Trials

Clinical trials were conducted to evaluate the immunogenicity,protective efficacy and safety of the vaccine NmVac A, C, Y & W-135 DTconjugate vaccine, against meningococcal infection, compared to AventisPasture Menactra A, C, Y and W-135 DT conjugate vaccine.

The two assay techniques used in the study were ELISA to detect antimeningococcal antibodies and serum bactericidal assay (the prevalence ofmeningococcal disease is inversely proportional to the polysaccharidespecific bactericidal titre in the serum)

ELISA

Noncompetitive ELISAs used to detect anti meningococcal antibodiesinvolve: 1) coating of a microtiter plate with the antigen to bestudied; 2) blocking of unbound sites on the plate with animmunologically neutral protein; 3) addition of test sera and specificbinding of antibodies to the solid-phase antigen on the plate; 4)addition of a detector antibody that recognizes the class or subclass ofserum antibody; 5) generation of a color change on the ELISA platelinked to the amount of bound detector antibody; 6) calculation ofconcentration of specific antibodies in test sample.

Principle of Serum Bactericidal Assay

N. Meningitidis target strains are lysed in the presence ofmeningococcal specific antibody and complement (antibody mediatedcomplement dependent killing). Serial dilutions of human sera areincubated with appropriate target strains and complement. Meningococcalspecific antibody binds to the target cell surface via meningococcalspecific protein or carbohydrate moieties. The serum bactericidal titerfor each unknown serum is expressed as the reciprocal serum dilutionyielding 50% killing as compared to the number of target cells presentbefore incubation with serum and complement.

Study Design

The study was a double blinded randomized controlled single armexperiment in Africa to evaluate the safety, protective efficacy andimmunogenicity of quadrivalent meningococcal meningitis vaccine (NmVacA, C, Y & W-135 DT conjugate vaccine) in healthy subjects using anestablished product in the market Menactra A, C, Y and W-135 DTconjugate as a control.

Vaccine Administration and Sampling

The trial was conducted on 101 volunteers between the ages of 13-30years of both genders fulfilling the eligibility criteria in city ofBouake in Ivory Coast. The Participants were coded, randomized and givena single dose of the coded vaccine. Both vaccines were administeredintramuscularly in the deltoid region of the arm.

NmVac A, C, Y, W-135 DT conjugate vaccine is a sterile, clear toslightly turbid liquid, composed of Neisseria Meningitidis serogroups A,C, Y and W-135, purified capsular polysaccharides antigens, each of themconjugated to diphtheria toxoid protein. Each 0.5 ml dose of the vaccineis formulated in sodium phosphate buffered isotonic sodium chloridesolution to contain 4 μg each of meningococcal A, C, Y and W-135polysaccharides conjugated to approximately 48 μg of diphtheria toxoidprotein as a carrier. It also contains Lactose as a stabilizer.

Blood samples were obtained 2 weeks pre-vaccination, 2 weeks and 8 weekspost-vaccination. Antibody response was determined using SerumBactericidal Antibody assay. All the samples were reported at PasteurInstitute, Ivory Coast. Serum Bactericidal Antibody titres of 128 andabove were taken to be protective and of positive seroconversion.

Results

The clinical study conducted to evaluate the immunogenicity, protectiveefficacy and safety of a new vaccine NmVac A, C, Y and W-135 DTconjugate against meningococcal infection, in volunteers 13-30 years ofage, both male and female, in the city of Bouake in Ivory Coast,compared to Aventis Pasture Menactra A, C, Y and W-135 DT conjugatevaccine, presently in the market showed the following results.

The primary objective was to evaluate the immunogenic response to NmVacA, C, Y and W-135 DT conjugate vaccine, compared to Aventis PastureMenactra A, C, Y and W-135 DT conjugate vaccine. This was done byestimating the Serum Bactericidal Antibody Titres at 2 and 8 weekspost-vaccination. Titres above 128 were taken to be positive forseroconversion and protective efficacy.

The primary hypothesis to show that NmVac A, C, Y and W-135 DT conjugatevaccine, is non inferior to Aventis Pasture Menactra A, C, Y and W-135DT conjugate vaccine was achieved for serogroup A. The upper limit ofthe one-sided 95% confidence interval of the difference of theproportion of subjects achieving seroconversion for the two vaccinegroups is less than 0.10. The upper limit of the two-sided 95%confidence interval is also evaluated and found to be less than 0.1 inaccordance with the current preferences of the United States Center forBiologics Evaluation and Research (CBER) for testing non-inferiorityimmunogenicity hypotheses. For the other Serogroups C, Y, and W-135, theupper limit of the one-sided 95% confidence interval of the differenceof the proportion of subjects achieving seroconversion for the twovaccine groups is slightly above 0.1.

The secondary objective was to evaluate the safety of the NmVac A, C, Yand W-135 DT conjugate vaccine, compared to Menactra A, C, Y and W-135DT conjugate vaccine in the population of City of Bouake. This was doneby monitoring for immediate reactions 15 minutes post-vaccination andthe local and systemic reactions were noted in the case report formbefore discharge. Further the participants were contacted by the medicalofficer at 24 hours, 48 hours and 72 hours for pre-specified adverseevents which included local reactions (pain, erythema, swelling andinduration) and systemic reactions (fever, rash, headache, photophobia,weakness, myalgia, arthralgia, nausea, vomiting, abdominal pain,diarrhea). Adverse events were also monitored at the subsequent Visitsat week 2 and week 8. There were no immediate reactions or seriousadverse events in participants of both vaccine groups.

The overall local reactions were fewer in the NmVac A, C, Y and W-135 DTconjugate vaccine recipients, compared to Menactra A, C, Y and W-135 DTconjugate vaccine recipients. The most common local reaction was pain atthe vaccine administration site, followed by swelling/induration. Nosystemic reactions were reported in the NmVac A, C, Y and W-135 DTconjugate vaccine recipients, whereas in the Menactra A, C, Y and W-135DT conjugate vaccine recipients the most common systemic adversereaction was headache (3.85%), followed by nausea (1.92%).

The phase III clinical trial report details that the NmVac A, C, Y andW-135 DT conjugate vaccine is non inferior to Menactra A, C, Y and W-135DT conjugate vaccine with regards to safety of the vaccine, also thestudy demonstrates a better safety profile for the new vaccine. Theimmunogenicity of the vaccine is non inferior to Menactra partially at 8weeks.

The invention claimed is:
 1. A modified Neisseria meningitidisfastidious culture medium (NMFM) for producing a higher amount ofcapsular polysaccharides and a lesser amount of cellular biomass fromNeisseria meningitidis comprising: DI (deionized) water, NaCl, K₂HPO₄,NH₄Cl, K₂SO₄, MgCl₂.6H₂O, CaCl₂.2H₂O, Glucose, L-Glutamic Acid,L-Arginine, Glycine, L-Serine, L-Cysteine.HCl, Fe (111) Citrate, YeastExtract (30 k dia-filtered), and antifoam, with the pH of thecomposition maintained from 6.5 to 7.0 for serogroups A, C, Y and W-135and characterized in that the use of the medium yields a higherpercentage of polysaccharides and a lower percentage of cellular biomassas compared to Catlin or Watson-Scherp medium.
 2. The culture mediumaccording to claim 1, a liter comprising: 800 mL of DI (deionized)water, NaCl—5.60 g, K₂HPO₄—4.10 g, NH₄Cl—1.00 g, K₂SO₄—0.90 g,MgCl₂-6H₂O—0.35 g, CaCl₂.2H₂O—0.03 g, Glucose—10.0 g, L-GlutamicAcid—3.80 g, L-Arginine—0.10 g, Glycine—0.20 g, L-Serine—0.50 g,L-Cysteine-HCl—0.10 g, Fe (111) Citrate—0.04 g, Yeast Extract (30 kdia-filtered)—0.1%, and antifoam-0.1 ml, with the pH of the compositionmaintained from 6.5 to 7.0 for serogroups A, C, Y and W-135, where themedium yields a higher percentage of polysaccharides and a lowerpercentage of cellular biomass as compared to Catlin or Watson-Scherpmedium.